Novel monobody therapy for pancreatic cancer Abstract The current work is based on the hypothesis that harnessing specific, basic elements of TCR/CD3 signaling can enhance anti-cancer T cell responses. T cell stimulation by antigen induces a conformational change in the CD3 complex associated with the TCR (CD3?c), the earliest molecular event marking productive TCR/CD3 triggering. CD3?c is necessary but not sufficient for optimal CD3 signaling, which additionally requires receptor cross-linking. As a consequence of CD3?c, a cryptic proline-rich sequence (PRS) in the cytoplasmic domain of CD3? becomes exposed to interact with adaptor proteins like Nck that contribute to CD3 signaling. Generation of CD3?c only results from exposure to strong antigens, but not to poorly immunogenic antigens, like most tumor associated antigens (TAAs). We recently discovered that a monovalent Fab fragment (Mono-Fab) that binds CD3 can induce CD3?c without inducing receptor cross-linking and intrinsic signal transduction. Such a Mono-Fab can enhance T cell responses to weak antigens in vitro and in vivo, an effect we call T cell ?co- potentiation?. Mono-Fabs combined with checkpoint inhibition stimulate remarkable anti-tumor responses in mouse models. Fabs suffer from short serum half-life and production issues making them less than ideal for clinical development. To address these problems we will construct a monobody form of a humanized anti-CD3? antibody. During this Phase I project, we will rank-order a panel of humanized anti-human CD3 monobodies and evaluate their activity in vitro and in an animal model of pancreatic cancer both alone and in combination with anti-PD-1. In Phase II, the lead construct and a back-up will be further evaluated in tumor models followed by IND-enabling studies to support clinical development of this novel therapeutic modality. A successful outcome of this work will provide a novel cancer therapy suitable for combination with chemotherapies and other immunotherapeutic agents, and likely to enhance many other approaches.